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Transcriptional control is exerted primarily through the binding of transcription factor proteins to regulatory elements in DNA. By virtue of eukaryotic DNA being complexed with histones, transcription factor binding to DNA alters or eliminates histone-DNA contacts, leading to increased accessibility of the DNA region to nuclease enzymes. This hypersensitivity to nuclease digestion has been used to define DNA binding events and regulatory elements across genomes, and to compare these attributes between cell types or conditions. These approaches make it possible to define the regulatory elements in a genome as well as to predict the regulatory networks of transcription factors and their target genes in a given cell state. As these chromatin accessibility assays are increasingly used, it is important to consider how to analyze the resulting data to avoid artifactual results or misinterpretation. In this review, we focus on some of the key technical and computational caveats associated with plant chromatin accessibility data, including strategies for sample preparation, sequencing, read mapping, and downstream analyses.

Here, Zhou et al. took advantage of Saccharomyces cerevisiae and its well-characterized mating and invasion pathways to explain how transcription factor variants alter motif recognition and gene expression, and ultimately, organismal phenotypes..., Amino acid substitutions are commonly found in human transcription factors, yet the functional consequences of much of this variation remain unknown, even in well-characterized DNA-binding domains. Here, we examine how six single-amino acid variants in the DNA-binding domain of Ste12—a yeast transcription factor regulating mating and invasion—alter Ste12 genome binding, motif recognition, and gene expression to yield markedly different phenotypes. Using a combination of the “calling-card” method, RNA sequencing, and HT-SELEX (high throughput systematic evolution of ligands by exponential enrichment), we find that variants with dissimilar binding and expression profiles can converge onto similar cellular behaviors. Mating-defective variants led to decreased expression of distinct subsets of genes necessary for mating. Hyper-invasive variants also decreased expression of subsets of genes involved in mating, but increased the expression of other subsets of genes associated with the cellular response to osmotic stress. While single-amino acid changes in the coding region of this transcription factor result in complex regulatory reconfiguration, the major phenotypic consequences for the cell appear to depend on changes in the expression of a small number of genes with related functions.

The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of polysaccharides. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation (4 and 7 DPA), enriching for seed coat cells with the INTACT method. We found over 3,000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with 7-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, plant regulatory landscapes from different tissues, cell types, or developmental stages were more dynamic than those generated from bulk tissue in response to environmental perturbations, highlighting the importance of extending studies of regulatory DNA to single tissues and cell types during development.

Single-cell RNA-seq can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach toA. thalianaroot cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single-cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single-cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.

The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of pectin. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation, enriching for seed coat cells with the INTACT method. We found over 3000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with seven-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, in this comparison, development exerted a stronger effect on the plant regulatory landscape than extreme environmental perturbations, highlighting the importance of extending studies of regulatory landscapes to other tissues and cell types during development.

Understanding gene regulatory networks in plants requires knowledge of cis- regulatory DNA, trans -acting factors, and their dynamics across development and in response to stimuli. Active cis -regulatory elements are hypersensitive to cleavage by the endonuclease DNase I. Motifs within DNase I hypersensitive sites indicate potential trans -acting factor occupancy and, when combined with DNase I cleavage data, can be used to construct provisional regulatory networks. Several recent studies have applied genome-wide DNase I hypersensitivity mapping to Arabidopsis thaliana and rice, generating chromatin accessibility landscapes for an array of tissues, cell types, and treatments. Here, we discuss these studies, with an emphasis on building regulatory networks and possible directions for improvements.

Cilia and flagella are widespread eukaryotic subcellular components that are conserved from green algae to mammals. In different organisms they function in cell motility, movement of extracellular fluids and sensory reception. While the function and structural description of cilia and flagella are well established, there are many questions that remain unanswered. In particular, very little is known about the developmental mechanisms by which cilia are generated and shaped and how their components are assembled into functional machineries. To find genes involved in cilia development we used as a search tool a promoter motif, the X-box, which participates in the regulation of certain ciliary genes in the nematode Caenorhabditis elegans. By using a genome search approach for X-box promoter motif-containing genes(xbx genes) we identified a list of about 750 xbx genes(candidates). This list comprises some already known ciliary genes as well as new genes, many of which we hypothesize to be important for cilium structure and function. We derived a C. elegans X-box consensus sequence by in vivo expression analysis. We found that xbx gene expression patterns were dependent on particular X-box nucleotide compositions and the distance from the respective gene start. We propose a model where DAF-19, the RFX-type transcription factor binding to the X-box, is responsible for the development of a ciliary module in C. elegans, which includes genes for cilium structure, transport machinery, receptors and other factors.

Summary Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis -regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis -regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.